Target substance detection device and target substance detection method

Abstract

A target substance detection device 1 comprises a target-substance-including base material 10 and a sensor body 20. The target-substance-including base material 10 includes: a base material 12, to the surface of which is immobilized at least some of a first capture molecule 11A; a target substance 13 which is bonded to the first capture molecule 11A; and an enzyme labelling second capture molecule 15 in which a second capture molecule 11B and an enzyme 14 are bonded, and in which the second capture molecule 11B is bonded to the target substance 13. The sensor body 20 includes an evaluation liquid 21 which contains a substrate 24 modified with the enzyme 14, a container 22 which accommodates the evaluation liquid 21, and an electric sensor 23 which is provided so as to contact the evaluation liquid 21 for sensing a component in the evaluation liquid 21. The target-substance-including base material 10 is separated and independent from the sensor body 20. The site at which the first capture molecule 11A of the target-substance-including base material 10 is immobilized to the surface of the base material 12 is immersed in the evaluation liquid 21.

Description

Target substance detection device and target substance detection method

[0001] The present invention relates to a target substance detection device and a target substance detection method.

[0002] There is a demand for a highly sensitive detection device and detection method that can rapidly detect target substances such as viruses and proteins.

[0003] Patent Document 1 discloses a method for measuring an electrical change for detecting or quantifying a target substance in a sample, the method comprising: 1) a step of binding a compound A capable of selectively binding to the target substance and generating a charged substance to the target substance; 2) a step of generating a charged substance in compound A; and 3) a step of measuring an electrical change caused by the charged substance in a required volume using a field-effect transistor.

[0004] In the method shown in FIG. 1 of Patent Document 1, a target substance is first immobilized on a field-effect transistor (FET). Compound A is urease modified at a selective binding site, and compound A generates ammonia in an aqueous reaction field containing a specific urea. This ammonia becomes ammonium ions, a charged substance, in water, and the generation of these ammonium ions is detected by the FET. The amount of ammonium ions can then be quantified based on the detected current. Because the amount of ammonium ions generated is governed by the amount of urease and the amount of urea introduced, the reaction rate between urease and urea is measured in advance, and the amount of selectively bound urease can be determined by tracking the change in the detected electrical quantity over a specified time period. The amount of selectively bound urease is proportional to the amount of target substance, thereby enabling the target substance to be quantified.

[0005] Patent Document 2 discloses a measuring device comprising: a container having an antibody against a substance to be measured; a sample solution supplying means for supplying a sample solution containing the substance to be measured to the container; an enzyme-labeled antibody supplying means for supplying an enzyme-labeled antibody in which an enzyme that generates a thiol compound and an antibody against the substance to be measured are bound to the container; a substrate supplying means for supplying a substrate for the enzyme; a field-effect transistor; an electrode connected to the gate of the field-effect transistor by wiring and in contact with the solution in the container; a reference electrode in contact with the solution in the container; a power source for applying a voltage between the electrode and the reference electrode; and a detection unit for detecting the output of the field-effect transistor.

[0006] In the method shown in Figure 1 of Patent Document 2, a sample solution is first injected into a reaction solution in a measurement cell using a sample solution injector, causing the antigen and antibody in the sample solution to bind. After a certain period of time, an enzyme-labeled antibody solution is injected into the reaction solution using an enzyme-labeled antibody solution injector, causing an antigen-antibody reaction and forming an antibody-antigen-enzyme-labeled antibody. The bound enzyme-labeled antibody and free enzyme-labeled antibody are then separated by washing the measurement cell and replacing the reaction solution in the measurement cell. After washing and replacing the solution in the measurement cell, a substrate for the labeled enzyme is injected using a substrate solution injector. The substrate is decomposed by the enzyme and a thiol compound is generated. The generated thiol compound adsorbs to a gold electrode formed on an insulated gate field-effect transistor (FET) and forms a self-assembled monolayer. As a result, the potential on the gold electrode changes. Measurements are performed by monitoring in real time the source-drain current in the insulated gate field-effect transistor, which changes before and after substrate injection using the substrate solution injector, and recording it using a signal processing circuit and a data processing device. The rate at which the thiol compound is adsorbed onto the gold electrode is proportional to the rate at which the thiol compound is produced, i.e., the amount of antibody-antigen-enzyme-labeled antibody. Therefore, by measuring the rate at which the thiol compound is adsorbed onto the gold electrode, the amount of the bound labeled enzyme, i.e., the amount of antigen in the sample solution, can be obtained. During measurement, a high-frequency voltage is applied to the reference electrode from a power source to reduce the influence of external fluctuations in the measurement.

[0007] JP 2018-36154 A JP 2007-263914 A

[0008] The method described in Patent Document 1 is characterized in that, rather than detecting the target substance itself, a compound A is utilized that selectively binds to the target substance and generates a charged substance through a predetermined reaction, and the charged substance generated from the compound A is detected. Patent Document 1 describes, as a preferred example of compound A, a substance having a portion that functions as an enzyme and a portion that selectively binds to the target substance (for example, an antibody modified with urease). In this case, ammonium ions generated by the reaction between urease and urea are detected.

[0009] However, the method described in Patent Document 1 requires the introduction of a substrate such as urea to react with an enzyme such as urease. Therefore, there is a risk that noise signals other than ammonium ions may be detected due to solution manipulation when introducing the substrate. Furthermore, to remove enzymes that are not bound to the target substance, it is necessary to perform operations such as washing the inside of the container or adding new solution to the container before introducing the substrate. As a result, the overall process may become complicated.

[0010] Although the method described in Patent Document 2 uses different types of enzyme and substrate, it does not detect the target substance itself but rather the reaction product produced by the enzyme reaction, just like the method described in Patent Document 1.

[0011] However, even in the method described in Patent Document 2, there is a risk of detecting noise signals due to solution manipulation when injecting the enzyme substrate. Furthermore, since it is necessary to perform operations such as cleaning the inside of the measurement cell and replacing the solution in the cell before injecting the substrate, the entire process may become complicated.

[0012] The present invention has been made to solve the above problems, and aims to provide a target substance detection device capable of suppressing noise signals, and a target substance detection method capable of suppressing noise signals.

[0013] The target substance detection device of the present invention includes a target substance-attached substrate and a sensor body. The target substance-attached substrate includes a substrate having first capture molecules immobilized on at least a portion of its surface, a target substance bound to the first capture molecules, and an enzyme-labeled second capture molecule in which a second capture molecule is bound to an enzyme and the second capture molecule is bound to the target substance. The sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for containing the evaluation liquid, and an electrical sensor arranged to contact the evaluation liquid for sensing components in the evaluation liquid. The target substance-attached substrate is independently separated from the sensor body. The portion of the target substance-attached substrate where the first capture molecules are immobilized on the surface of the substrate is immersed in the evaluation liquid.

[0014] The method for detecting a target substance of the present invention comprises the steps of: preparing a substrate with a target substance, the substrate including a substrate having a first capture molecule immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule in which a second capture molecule is bound to an enzyme and the second capture molecule is bound to the target substance; immersing the portion of the substrate with the target substance where the first capture molecule is immobilized on the surface of the substrate in an evaluation liquid containing a substrate to be modified by the enzyme; and sensing components in the evaluation liquid using an electrical sensor arranged to come into contact with the evaluation liquid.

[0015] According to the present invention, it is possible to provide a target substance detection device capable of suppressing noise signals. Furthermore, according to the present invention, it is possible to provide a target substance detection method capable of suppressing noise signals.

[0016] Fig. 1 is a schematic diagram showing an example of a target substance detection device of the present invention. Fig. 2 is a schematic diagram showing a state in which a target substance-attached substrate and a sensor main body constituting the target substance detection device shown in Fig. 1 are separated. Fig. 3 shows the gate-source voltage V ｇｓ and the source-drain current I ｄｓ4 is a graph showing an example of the relationship between the charge neutral point CNP and the target substance. FIG. 4 is a graph showing an example of the change over time of the charge neutral point CNP. FIG. 5 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 1. FIG. 6 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 2. FIG. 7 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 3. FIG. 8 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 4. FIG. 9 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 5. FIG. 10 is a schematic diagram showing a target substance detection device and a target substance detection method according to Example 6. FIG. 11 is a schematic diagram showing another example of a target substance-attached substrate.

[0017] The target substance detection device and target substance detection method of the present invention will be described below. Note that the present invention is not limited to the following configurations and may be modified as appropriate within the scope of the present invention. Furthermore, a combination of multiple individual preferred configurations described below also constitutes the present invention.

[0018] The drawings shown below are schematic diagrams, and the dimensions, aspect ratio, scale, etc. may differ from those of the actual product.

[0019] FIG. 1 is a schematic diagram showing an example of a target substance detection device of the present invention.

[0020] 1 includes a target substance-attached substrate 10 and a sensor body 20. The target substance-attached substrate 10 is independent and separate from the sensor body 20.

[0021] FIG. 2 is a schematic diagram showing the target substance-carrying substrate and the sensor main body that constitute the target substance detection device shown in FIG. 1 in a separated state.

[0022] 1 and 2 , the target substance-attached substrate 10 includes a substrate 12 having first capturing molecules 11A immobilized on at least a portion of its surface, a target substance 13 bound to the first capturing molecules 11A, and an enzyme-labeled second capturing molecule 15 in which a second capturing molecule 11B is bound to an enzyme 14 and the second capturing molecule 11B is bound to the target substance 13. The target substance-attached substrate 10 has a structure in which the target substance 13 is sandwiched between the first capturing molecule 11A and the enzyme-labeled second capturing molecule 15.

[0023] The first capturing molecule 11A is a molecule capable of capturing the target substance 13. Examples of the first capturing molecule 11A include an antibody, an aptamer, a peptide, a sugar chain, a receptor, a molecular template, an antigen for an antibody, and a secondary antibody for an antibody. The first capturing molecule 11A may be fixed or adhered to the surface of the substrate 12 as long as it remains on the surface of the substrate 12, or may be movable with a certain degree of freedom.

[0024] The material of the substrate 12 is not particularly limited, and examples thereof include glass, resin, ceramic, metal, etc. The size, thickness, shape, etc. of the substrate 12 are also not particularly limited, but it is preferable that the surface include an uneven structure so as to increase the surface area.

[0025] Examples of the target substance 13 include viruses, proteins, nucleic acids, and low molecular weight biological substances.

[0026] The second capturing molecule 11B is bound to the enzyme 14 to form an enzyme-labeled second capturing molecule 15. The second capturing molecule 11B is a molecule capable of capturing the target substance 13. Examples of the second capturing molecule 11B include antibodies, aptamers, peptides, sugar chains, receptors, molecular templates, and secondary antibodies against antibodies. The second capturing molecule 11B may be the same type as the first capturing molecule 11A, or may be a different type.

[0027] Examples of the enzyme 14 include urease and L-amino acid oxidase, but are not limited to these as long as they are enzymes that produce charged substances.

[0028] As shown in Figures 1 and 2, the sensor body 20 includes an evaluation liquid 21 containing a substrate 24 to be modified by the enzyme 14, a container 22 for accommodating the evaluation liquid 21, and an electrical sensor 23 arranged to come into contact with the evaluation liquid 21 for sensing the components in the evaluation liquid 21.

[0029] The electrical sensor 23 is different from both an optical sensor and a chemical sensor. In the example shown in Figures 1 and 2, the electrical sensor 23 is a field effect transistor (FET) sensor whose semiconductor channel layer is made of graphene.

[0030] Although not shown in FIGS. 1 and 2, the sensor body 20 may be provided with a plurality of electrical sensors 23 .

[0031] 1 and 2 , the electrical sensor 23 includes a semiconductor layer 25, and a source electrode 26 and a drain electrode 27 electrically connected to the semiconductor layer 25. The semiconductor layer 25 between the source electrode 26 and the drain electrode 27 forms a channel of the electrical sensor 23.

[0032] 1 and 2, the electrical sensor 23 may further include a gate electrode for applying an electric field from the outside to the semiconductor layer 25. In this case, the gate electrode is immersed in the evaluation liquid 21.

[0033] The number of layers in the semiconductor layer 25 is not limited to one, and may be two or three or more. The number of layers in the semiconductor layer 25 is preferably 10 or less, and more preferably 5 or less. The number of layers does not need to be uniform throughout the semiconductor layer 25; for example, one layer portion and two or more layers portion may be mixed. The number of layers in the semiconductor layer 25 can be measured, for example, by Raman spectroscopy or cross-sectional observation using a transmission electron microscope (TEM).

[0034] 1 and 2 , the source electrode 26 and the drain electrode 27 are disposed on an insulating substrate 28 at a distance from each other, and the insulating substrate 28 is exposed between the source electrode 26 and the drain electrode 27. The semiconductor layer 25 is disposed on the insulating substrate 28 so as to cover the exposed portion of the insulating substrate 28. The semiconductor layer 25 may be disposed so as to cover the end of the source electrode 26 or the end of the drain electrode 27.

[0035] The source electrode 26 and the drain electrode 27 are electrodes with a multilayer structure in which, for example, a titanium (Ti) layer and a gold (Au) layer are stacked. As the electrode material, in addition to titanium and gold, metals such as gold, platinum, titanium, and palladium may be used in a single layer, or two or more metals may be combined to form a multilayer structure.

[0036] When the electrical sensor 23 includes a gate electrode, the gate electrode applies a potential to the source electrode 26 and the drain electrode 27, and generally uses a reference electrode such as a silver-silver chloride electrode or a noble metal. The gate electrode is provided at a location other than the location where the source electrode 26 and the drain electrode 27 are formed. The gate electrode is usually provided on the insulating substrate 28 or at a location other than the insulating substrate 28, but is preferably provided above the source electrode 26 or the drain electrode 27.

[0037] The evaluation solution 21 contains a substrate 24 that is modified by the enzyme 14. The evaluation solution 21 is, for example, an electrolytic solution containing an electrolyte.

[0038] Examples of the substrate 24 include urea and L-amino acids.

[0039] The container 22 includes, for example, an insulating substrate 28 and a partition wall 29 provided on the insulating substrate 28 .

[0040] 1 and 2, the insulating substrate 28 is, for example, a silicon oxide (SiO ２Examples of the insulating substrate 28 include a thermally oxidized silicon substrate on which a thermally oxidized silicon (Si) layer is formed, a boron nitride (BN) substrate, etc. The material of the insulating substrate 28 is not particularly limited, and examples thereof include inorganic compounds such as silicon oxide, silicon nitride, aluminum oxide, titanium oxide, and calcium fluoride, and organic compounds such as acrylic resin, polyimide, and fluororesin. The shape of the insulating substrate 28 is not particularly limited, and may be a flat plate or a curved plate. The insulating substrate 28 may be flexible.

[0041] Examples of the partition wall 29 include, but are not limited to, silicone plates also called rubber pools, epoxy resins, acrylic resins, and the like.

[0042] 2, the target substance-attached substrate 10 is immersed in the evaluation liquid 21 of the sensor body 20. As a result, in the target substance detection device 1 shown in FIG. 1, the portion of the target substance-attached substrate 10 where the first capturing molecules 11A are immobilized on the surface of the substrate 12 is immersed in the evaluation liquid 21.

[0043] An example of the method for detecting a target substance of the present invention will now be described with reference to FIGS.

[0044] First, a target substance-attached substrate 10 is prepared. As described above, the target substance-attached substrate 10 includes a substrate 12 having first capturing molecules 11A immobilized on at least a portion of its surface, a target substance 13 bound to the first capturing molecules 11A, and an enzyme-labeled second capturing molecule 15 in which second capturing molecules 11B are bound to an enzyme 14 and the second capturing molecules 11B are bound to the target substance 13.

[0045] Next, the target substance-attached substrate 10 is immersed in an evaluation liquid 21 containing a substrate 24 at a portion where the first capturing molecules 11A are immobilized on the surface of the substrate 12.

[0046] The substrate 24 is modified by the enzyme 14. For example, when the substrate 24 is urea and the enzyme 14 is urease, ammonia and carbon dioxide are generated by the reaction of the urea with water. The ammonia and carbon dioxide exist as ammonium ions and carbonate ions in the water. Therefore, the total amount of ions in the evaluation liquid 21 changes before and after the target substance-attached substrate 10 is immersed in the evaluation liquid 21.

[0047] Therefore, the components in the evaluation liquid 21 are sensed using an electrical sensor 23 provided so as to be in contact with the evaluation liquid 21. For example, by observing changes in the components in the evaluation liquid 21 before and after immersing the target substance-attached substrate 10 in the evaluation liquid 21, it is possible to sense the presence or concentration of a reaction product produced by an enzyme reaction. Because the amount of the reaction product is proportional to the amount of the target substance 13, it is possible to sense the presence or concentration of the target substance 13 as a result.

[0048] As an example, when the electrical sensor 23 is an FET sensor, a voltage is first applied between the source electrode 26 and the drain electrode 27 to generate a current (hereinafter referred to as a source-drain current I ｄｓ The value of this parameter is measured.

[0049] While the voltage applied between the source electrode 26 and the drain electrode 27 is kept constant, the voltage applied between the gate electrode (not shown) and the source electrode 26 (hereinafter referred to as the gate-source voltage V ｇｓ ) is repeatedly swept to measure the source-drain current I ｄｓ The gate-source voltage V when is minimum ｇｓ In the following description, the source-drain current I ｄｓ The gate-source voltage V when is minimum ｇｓ The value of this is called the charge neutrality point (CNP).

[0050] FIG. 3 shows the gate-source voltage V ｇｓ and the source-drain current I ｄｓ 10 is a graph showing an example of the relationship between

[0051] For example, when the total amount of ions in the evaluation liquid 21 changes before and after the target substance-attached substrate 10 is immersed in the evaluation liquid 21, as shown in FIG. ｄｓ The gate-source voltage V when is minimum ｇｓ , i.e., the charge neutral point CNP, changes.

[0052] FIG. 4 is a graph showing an example of the change in the charge neutral point CNP over time.

[0053] In Figure 4, the change in the charge neutral point CNP over time when the target substance-containing substrate 10 is immersed in the evaluation solution 21 containing the substrate 24 is indicated by a circle (positive control), and the change in the charge neutral point CNP over time when a substrate to which the first capturing molecule 11A is not immobilized is immersed is indicated by a square (negative control). As indicated by the arrows in Figure 4, the charge neutral point CNP changes before and after the target substance-containing substrate 10 is immersed in the evaluation solution 21. The change in the charge neutral point CNP corresponds to the amount of reaction product produced by the enzyme reaction. Therefore, by evaluating the change in the charge neutral point CNP, the target substance 13 can be detected.

[0054] As described above, the target substance detection device and target substance detection method of the present invention detect the reaction product produced by the enzyme reaction, rather than the target substance itself, as in Patent Documents 1 and 2. Therefore, any target substance can be detected regardless of the presence or absence of a charge on the target substance, its size, the size of the target substance, etc.

[0055] On the other hand, in the target substance detection device and target substance detection method of the present invention, unlike Patent Documents 1 and 2, a substrate to be modified by an enzyme is not introduced, but rather a substrate with a target substance is immersed in an evaluation solution containing the substrate in advance. This prevents the solute components contained in the evaluation solution from changing before and after the start of sensing, thereby suppressing noise signals caused by such changes. Furthermore, operations such as changing or cleaning the solution to introduce the substrate, or adding a solution, are not required.

[0056] In the target substance detection device of the present invention, it is preferable that a portion of the target substance-bearing substrate protrudes from the evaluation liquid. Similarly, in the target substance detection method of the present invention, it is preferable that the target substance-bearing substrate is immersed in the evaluation liquid so that a portion of the target substance-bearing substrate protrudes from the evaluation liquid. Specifically, it is preferable that a portion of the substrate where the first capturing molecules are not immobilized protrudes from the evaluation liquid. Since the area of ​​the portion where the first capturing molecules are immobilized on the surface of the substrate can be controlled, the dynamic range of the sensor can be improved. Furthermore, since the location where the target substance-bearing substrate is immersed in the evaluation liquid can be controlled, for example, the portion where the first capturing molecules are immobilized on the surface of the substrate can be positioned near the sensing section, or near the semiconductor layer section in FIG. 1.

[0057] In the target substance detection device and target substance detection method of the present invention, for example, the substrate is urea and the enzyme is urease. When non-ionic urea is hydrolyzed, it is decomposed into ionic ammonia and carbon dioxide, and therefore the target substance can be detected by evaluating the ionic components in the evaluation solution.

[0058] In the target substance detection device and the target substance detection method of the present invention, the electrical sensor is preferably any one of an FET sensor, a conductivity sensor, an ion electrode, and an impedance sensor. Use of these electrical sensors enables highly sensitive evaluation of the components in the evaluation solution.

[0059] In the target substance detection device and target substance detection method of the present invention, the electrical sensor is preferably an FET sensor, and particularly preferably an FET sensor with graphene as the semiconductor channel.

[0060] Graphene is a two-dimensional material consisting of carbon atoms bonded in a hexagonal network, which has a very large specific surface area (surface area per volume) and very high electrical mobility.

[0061] In the target substance detection device and the target substance detection method of the present invention, when the electrical sensor is an FET sensor, the FET sensor may have a semiconductor channel made of a material other than graphene, such as carbon nanotubes, molybdenum disulfide, boron nitride, silicon, an oxide semiconductor, or an organic semiconductor.

[0062] In the target substance detection device and target substance detection method of the present invention, when the electrical sensor is an FET sensor, an ion-sensitive membrane may be provided on the surface of the FET sensor. The ion-sensitive membrane enables highly sensitive sensing of ion components.

[0063] Examples of materials for the ion-sensitive membrane include vinyl chloride containing ammonium ionophores, such as nonactin.

[0064] In the target substance detection device and target substance detection method of the present invention, the conductivity of the evaluation liquid before the target substance-bearing substrate is immersed is preferably 1 mS / cm or less. In this case, the rate of change before and after the target substance-bearing substrate is immersed in the evaluation liquid is large, allowing for highly sensitive sensing.

[0065] The conductivity of the evaluation liquid before the target substance-carrying substrate is immersed is, for example, 1 μS / cm or more.

[0066] In addition, if the conductivity of the evaluation liquid before the substrate with the target substance is immersed is 1 mS / cm or less, the conductivity of the evaluation liquid during sensing may be 1 mS / cm or less or may exceed 1 mS / cm.

[0067] In this specification, the conductivity of the evaluation liquid means the conductivity measured by an AC two-electrode method.

[0068] Hereinafter, examples will be given that more specifically disclose the target substance detection device and target substance detection method of the present invention, but the present invention is not limited to these examples.

[0069] Example 1 FIG. 5 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to Example 1. As shown in FIG.

[0070] In Example 1, the substrate 10 with the target substance is prepared by immersing a substrate 12 having first capture molecules 11A immobilized on at least a portion of its surface in a preparation liquid 30 containing enzyme-labeled second capture molecules 15 and a target substance 13, as shown in Figure 5.

[0071] For example, the preparation solution 30 can be prepared by supplying a sample containing the target substance 13 to an electrolyte solution containing the enzyme-labeled second capturing molecules 15. When the sample containing the target substance 13 is a liquid, the sample may be dropped using a dropper or the like, or the sample may be introduced using a flow path.

[0072] Examples of samples containing the target substance 13 include body fluids such as saliva, nasal swabs, throat swabs, tears, and blood of a subject, biological samples such as urine and feces, suspensions of cells or viruses themselves, drinking water, sewage, breath, etc. The sample containing the target substance 13 does not have to be a liquid.

[0073] After preparing the substrate 10 with the target substance, the portion of the substrate 10 with the target substance where the first capture molecule 11A is immobilized on the surface of the substrate 12 is immersed in an evaluation liquid 21 containing a substrate 24.

[0074] Second Embodiment FIG. 6 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to a second embodiment.

[0075] In Example 2, the substrate 10 with the target substance is prepared by immersing a substrate 12, on which first capture molecules 11A are immobilized on at least a portion of the surface and on which target substances 13 are bound, in a preparation liquid 30 containing enzyme-labeled second capture molecules 15, as shown in Figure 6.

[0076] For example, by supplying a sample containing a target substance 13 to a substrate 12 having a first capture molecule 11A immobilized on at least a portion of its surface, the target substance 13 can be bound to the first capture molecule 11A immobilized on the surface of the substrate 12.

[0077] After preparing the substrate 10 with the target substance, the portion of the substrate 10 with the target substance where the first capture molecule 11A is immobilized on the surface of the substrate 12 is immersed in an evaluation liquid 21 containing a substrate 24.

[0078] Third Embodiment FIG. 7 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to a third embodiment.

[0079] In Example 3, the substrate on which the first capturing molecule is immobilized is composed of a magnetic particle 31 on which the first capturing molecule 11A is immobilized and a magnetic substrate 32 that holds the magnetic particle 31 by magnetism, as shown in Figure 7.

[0080] By using the magnetic substrate 32 and the magnetic particles 31, it is possible to capture a larger amount of the target substance 13. As a result, it is possible to increase the sensitivity.

[0081] The method for binding the target substance 13 to the first capturing molecule 11A may be the same as that in the first embodiment or the second embodiment.

[0082] Examples of materials for the magnetic particles 31 include ferrite, iron oxide, iron, nickel, cobalt, etc. The average particle diameter of the magnetic particles 31 is, for example, 50 nm or more and 5 μm or less.

[0083] Examples of materials for the magnetic substrate 32 include ferrite, iron oxide, iron, nickel, cobalt, etc. The material for the magnetic substrate 32 may be the same as or different from the material for the magnetic particles 31.

[0084] Example 4 FIG. 8 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to Example 4. As shown in FIG.

[0085] In Example 4, as shown in FIG. 8, the container 22 is divided into a plurality of wells 33, each containing the evaluation liquid 21 in a volume of 1 pL or less.

[0086] Since the target substance 13 is confined in a microscopic space of well 33 and detected, the signal can be digitized and highly sensitively sensed by assigning a value of 1 to well 33 in which the target substance 13 is detected and a value of 0 to well 33 in which the target substance 13 is not detected.

[0087] The container 22 is preferably divided into 100 or more wells 33, more preferably into 1000 or more wells 33, and even more preferably into 10,000 or more wells 33. On the other hand, the container 22 is preferably divided into 1,000,000 or less wells 33, and even more preferably into 500,000 or less wells 33.

[0088] Each well 33 preferably contains the evaluation liquid 21 in a volume of 1 pL or less, more preferably 100 fL or less, whereas each well 33 preferably contains the evaluation liquid 21 in a volume of 1 fL or more.

[0089] The substrate 12 may be in the form of particles or beads, for example. In this case, the average particle size of the substrate 12 is, for example, 50 nm or more and 5 μm or less. The substrate 12 may be magnetic.

[0090] Fifth Embodiment FIG. 9 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to a fifth embodiment.

[0091] In Example 5, the end of the substrate 12 is divided into two or more parts as shown in Fig. 9. In the example shown in Fig. 9, the end of the substrate 12 is divided into two parts.

[0092] 9, a first capturing molecule 11A is immobilized on one end of the substrate 12, and no first capturing molecule 11A is immobilized on the other end of the substrate 12. The ends of the substrate 12 are immersed in different evaluation liquids 21.

[0093] In Example 5, the side on which the first capturing molecules 11A are not immobilized serves as a negative control, thereby reducing the possibility of false positives.

[0094] The number of ends to which the first capturing molecules 11A are immobilized may be two or more. Similarly, the number of ends to which the first capturing molecules 11A are not immobilized may be two or more. The number of ends to which the first capturing molecules 11A are not immobilized may be the same as or different from the number of ends to which the first capturing molecules 11A are immobilized.

[0095] The method for binding the target substance 13 to the first capturing molecule 11A may be the same as that in the first embodiment or the second embodiment.

[0096] Sixth Embodiment FIG. 10 is a schematic diagram illustrating a target substance detection device and a target substance detection method according to a sixth embodiment.

[0097] In Example 6, as shown in Fig. 10, the target material 13 includes a first target material 13A and a second target material 13B. Furthermore, the end of the substrate 12 is divided into two or more parts. In the example shown in Fig. 10, the end of the substrate 12 is divided into two parts.

[0098] 10, a first capturing molecule 11A is immobilized on one end of a substrate 12, and a third capturing molecule 11C, which has a different binding target from the first capturing molecule 11A, is immobilized on the other end of the substrate 12. A first target substance 13A binds to the first capturing molecule 11A, and a second target substance 13B binds to the third capturing molecule 11C.

[0099] The target substance-attached substrate 10 further includes an enzyme-labeled fourth capturing molecule 16 in which a fourth capturing molecule 11D is bound to an enzyme 14, and the fourth capturing molecule 11D is bound to a second target substance 13B. Each end of the substrate 12 is immersed in a separate evaluation liquid 21.

[0100] In the sixth embodiment, multiple types of target substances 13 can be detected simultaneously.

[0101] The number of ends to which the first capturing molecules 11A are immobilized may be two or more. Similarly, the number of ends to which the third capturing molecules 11C are immobilized may be two or more. The number of ends to which the third capturing molecules 11C are immobilized may be the same as or different from the number of ends to which the first capturing molecules 11A are immobilized.

[0102] The enzyme 14 bound to the fourth capturing molecule 11D may be the same as or different from the enzyme 14 bound to the second capturing molecule 11B. If the enzyme 14 bound to the fourth capturing molecule 11D is different from the enzyme 14 bound to the second capturing molecule 11B, the substrates 24 contained in the respective evaluation solutions 21 will also be different. The fourth capturing molecule 11D may be the same type as the third capturing molecule 11C, or a different type.

[0103] The method for binding the first target substance 13A to the first capturing molecule 11A may be the same as that in Example 1 or that in Example 2. The method for binding the second target substance 13B to the third capturing molecule 11C may be the same as that in Example 1 or that in Example 2.

[0104] In the example shown in FIG. 10, two types of target substances 13 are detected, but three or more types of target substances 13 may be detected.

[0105] As in Example 5, the substrate 12 may have an end portion to which the first capturing molecules 11A, the third capturing molecules 11C, etc. are not immobilized.

[0106] The target substance detection device and target substance detection method of the present invention are not limited to the above-mentioned embodiments and examples, and various applications and modifications can be made within the scope of the present invention regarding the configuration of the detection device, manufacturing conditions, etc.

[0107] For example, the enzyme-labeled second capturing molecule is formed by binding the second capturing molecule to an enzyme, and the second capturing molecule and the enzyme may be directly or indirectly bound to each other.Similarly, the enzyme-labeled fourth capturing molecule is formed by binding the fourth capturing molecule to an enzyme, and the fourth capturing molecule and the enzyme may be directly or indirectly bound to each other.

[0108] FIG. 11 is a schematic diagram showing another example of a substrate with a target substance.

[0109] The substrate 10A with target substance shown in Figure 11 includes a substrate 12 having a first capture molecule 11A immobilized on at least a portion of its surface, a target substance 13 bound to the first capture molecule 11A, and an enzyme-labeled second capture molecule 15A in which a second capture molecule 11B and an enzyme 14 are bonded and the second capture molecule 11B is bound to the target substance 13.

[0110] In the enzyme-labeled second capturing molecule 15A, the second capturing molecule 11B is indirectly bound to the enzyme 14. Specifically, the secondary capturing molecule 11b is bound to the enzyme 14, and the secondary capturing molecule 11b is bound to the second capturing molecule 11B, thereby forming the enzyme-labeled secondary capturing molecule 17, and the enzyme-labeled second capturing molecule 15A is formed by binding the enzyme-labeled secondary capturing molecule 17 to the second capturing molecule 11B.

[0111] In the target substance detection device and target substance detection method of the present invention, the structure in which the enzyme is immobilized on the substrate via the target substance may be a sandwich structure of (1) a first capture molecule immobilized on the substrate / (2) the target substance / (3) an enzyme-labeled second capture molecule, or may be a structure of (1) a first capture molecule immobilized on the substrate / (2) the target substance / (3) a second capture molecule / (4) an enzyme-labeled secondary capture molecule. The latter structure eliminates the need to prepare a new enzyme-labeled secondary capture molecule when the target substance changes, allowing the enzyme-labeled secondary capture molecule to be commonly used for various target substances. Furthermore, by making the enzyme-labeled secondary capture molecule a molecule that recognizes multiple sites on the second capture molecule, multiple enzyme-labeled capture molecules can be immobilized on the substrate. As a result, the amount of enzyme immobilized on the substrate increases, enabling the sensor to operate with high sensitivity. A typical example of such a secondary capture molecule is a polyclonal antibody.

[0112] The present specification discloses the following:

[0113] <1> A target substance detection device comprising: a substrate with a target substance; and a sensor body; wherein the substrate with the target substance includes a substrate having first capture molecules immobilized on at least a portion of its surface, a target substance bound to the first capture molecules, and an enzyme-labeled second capture molecule formed by binding a second capture molecule to an enzyme, the second capture molecule being bound to the target substance; and the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electrical sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; wherein the substrate with the target substance is independently separated from the sensor body; and wherein the portion of the substrate with the target substance where the first capture molecules are immobilized on the surface of the substrate is immersed in the evaluation liquid.

[0114] <2> The target substance detection device according to <1>, wherein a part of the target substance-attached substrate protrudes from the evaluation liquid.

[0115] <3> The device for detecting a target substance according to <1> or <2>, wherein the substrate is urea and the enzyme is urease.

[0116] <4> The target substance detection device according to any one of <1> to <3>, wherein the electrical sensor is any one of an FET sensor, a conductivity sensor, an ion electrode, and an impedance sensor.

[0117] <5> The target substance detection device according to any one of <1> to <4>, wherein the electrical sensor is an FET sensor using graphene as a semiconductor channel.

[0118] <6> The target substance detection device according to any one of <1> to <5>, wherein the electrical sensor is an FET sensor, and an ion-sensitive film is provided on the surface of the FET sensor.

[0119] <7> The target substance detection device according to any one of <1> to <6>, wherein the conductivity of the evaluation liquid before the target substance-bearing substrate is immersed is 1 mS / cm or less.

[0120] <8> The target substance detection device according to any one of <1> to <7>, wherein the substrate to which the first capture molecules are immobilized is composed of magnetic particles to which the first capture molecules are immobilized and a magnetic substrate that magnetically holds the magnetic particles.

[0121] <9> The target substance detection device according to any one of <1> and <3> to <7>, wherein the container is divided into a plurality of wells, each of which holds the evaluation liquid in a volume of 1 pL or less.

[0122] <10> The target substance detection device according to any one of <1> to <8>, wherein an end of the substrate is divided into two or more parts, the first capture molecule is immobilized to at least one end of the substrate, and the first capture molecule is not immobilized to at least one end of the substrate, and the ends of the substrate are immersed in different evaluation liquids.

[0123] <11> The target substance detection device according to any one of <1> to <8> and <10>, wherein the target substance includes a first target substance and a second target substance; an end of the substrate is divided into two or more parts; the first capturing molecule is immobilized to at least one end of the substrate; a third capturing molecule is immobilized to at least one end of the substrate, the third capturing molecule having a binding target different from that of the first capturing molecule; the first target substance is bound to the first capturing molecule; and the second target substance is bound to the third capturing molecule; the target substance-attached substrate further includes an enzyme-labeled fourth capturing molecule bound to a fourth capturing molecule, the fourth capturing molecule being bound to an enzyme, and the fourth capturing molecule is bound to the second target substance; and the ends of the substrate are immersed in different evaluation solutions.

[0124] <12> A method for detecting a target substance, comprising: a step of preparing a target substance-attached substrate, the target substance including a substrate having a first capture molecule immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule is bound to an enzyme and the second capture molecule is bound to the target substance; a step of immersing the target substance-attached substrate at a portion where the first capture molecule is immobilized on the surface of the substrate in an evaluation liquid containing a substrate to be modified by the enzyme; and a step of sensing components in the evaluation liquid using an electrical sensor provided so as to be in contact with the evaluation liquid.

[0125] <13> The method for detecting a target substance according to <12>, wherein the substrate carrying the target substance is immersed in the evaluation liquid so that a part of the substrate carrying the target substance protrudes from the evaluation liquid.

[0126] <14> The method for detecting a target substance according to <12> or <13>, wherein the substrate with the target substance is prepared by immersing the substrate, on at least a portion of whose surface the first capture molecule is immobilized, in a preparation solution containing the enzyme-labeled second capture molecule and the target substance.

[0127] <15> The method for detecting a target substance according to <12> or <13>, wherein the substrate with the target substance is prepared by immersing the substrate, in which the first capture molecule is immobilized on at least a portion of the surface thereof and the target substance is bound to the first capture molecule, in a preparation solution containing the enzyme-labeled second capture molecule.

[0128] <16> The method for detecting a target substance according to any one of <12> to <15>, wherein the substrate to which the first capture molecules are immobilized is composed of magnetic particles to which the first capture molecules are immobilized and a magnetic substrate that magnetically holds the magnetic particles.

[0129] <17> The method for detecting a target substance according to <12>, <14> or <15>, wherein the container is divided into a plurality of wells, each of which holds the evaluation liquid in a volume of 1 pL or less.

[0130] <18> The method for detecting a target substance according to any one of <12> to <16>, wherein an end of the substrate is divided into two or more parts, the first capturing molecule is immobilized to at least one end of the substrate, and the first capturing molecule is not immobilized to at least one end of the substrate, and the ends of the substrate are immersed in different evaluation solutions.

[0131] <19> The method for detecting a target substance according to any one of <12> to <16> and <18>, wherein the target substance includes a first target substance and a second target substance; an end of the substrate is divided into two or more parts; the first capturing molecule is immobilized to at least one end of the substrate; a third capturing molecule is immobilized to at least one end of the substrate, the third capturing molecule having a binding target different from that of the first capturing molecule; the first target substance is bound to the first capturing molecule; and the second target substance is bound to the third capturing molecule; the target substance-attached substrate further includes a fourth capturing molecule bound to an enzyme, the fourth capturing molecule being an enzyme-labeled fourth capturing molecule bound to the second target substance; and the ends of the substrate are immersed in different evaluation solutions.

[0132] <20> The method for detecting a target substance according to any one of <12> to <19>, wherein the electrical sensor is any one of an FET sensor, a conductivity sensor, an ion electrode, and an impedance sensor.

[0133] 1 Target substance detection device 10, 10A Substrate with target substance 11A First capture molecule 11B Second capture molecule 11C Third capture molecule 11D Fourth capture molecule 11b Secondary capture molecule 12 Substrate 13 Target substance 13A First target substance 13B Second target substance 14 Enzyme 15, 15A Enzyme-labeled second capture molecule 16 Enzyme-labeled fourth capture molecule 17 Enzyme-labeled secondary capture molecule 20 Sensor body 21 Evaluation liquid 22 Container 23 Electrical sensor 24 Substrate 25 Semiconductor layer 26 Source electrode 27 Drain electrode 28 Insulating substrate 29 Partition 30 Preparation liquid 31 Magnetic particles 32 Magnetic substrate 33 Well

Claims

1. A substrate having a target substance and a sensor body are provided, The target substance-attached substrate includes a substrate having a first capture molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule and an enzyme are bound to each other, and the second capture molecule is bound to the target substance; the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electric sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; the target substance-bearing substrate is independent and separate from the sensor body; a portion of the substrate having the target substance, at which the first capturing molecule is immobilized on the surface of the substrate, is immersed in the evaluation liquid; The container is divided into a plurality of wells, each of which holds the evaluation liquid in a volume of 1 pL or less.

2. A substrate having a target substance and a sensor body are provided, The target substance-attached substrate includes a substrate having a first capture molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule and an enzyme are bound to each other, and the second capture molecule is bound to the target substance; the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electric sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; the target substance-bearing substrate is independent and separate from the sensor body; a portion of the substrate having the target substance, at which the first capturing molecule is immobilized on the surface of the substrate, is immersed in the evaluation liquid; The end of the substrate is divided into two or more parts, The first capturing molecule is immobilized on at least one end of the substrate, The first capturing molecule is not immobilized on at least one end of the substrate, A target substance detection device, wherein each end of the substrate is immersed in a different evaluation liquid.

3. A substrate having a target substance and a sensor body are provided, The target substance-attached substrate includes a substrate having a first capture molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule and an enzyme are bound to each other, and the second capture molecule is bound to the target substance; the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electric sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; the target substance-bearing substrate is independent and separate from the sensor body; a portion of the substrate having the target substance, at which the first capturing molecule is immobilized on the surface of the substrate, is immersed in the evaluation liquid; The target substance includes a first target substance and a second target substance, The end of the substrate is divided into two or more parts, The first capturing molecule is immobilized on at least one end of the substrate, a third capturing molecule having a binding target different from that of the first capturing molecule is immobilized on at least one end of the substrate; The first target substance is bound to the first capturing molecule, The second target substance is bound to the third capturing molecule, the target substance-attached substrate further includes an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound to the second target substance, and the fourth capture molecule is bound to the second target substance; A target substance detection device, wherein each end of the substrate is immersed in a different evaluation liquid.

4. A substrate having a target substance and a sensor body are provided, The target substance-attached substrate includes a substrate having a first capture molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule and an enzyme are bound to each other, and the second capture molecule is bound to the target substance; the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electric sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; the target substance-bearing substrate is independent and separate from the sensor body; a portion of the substrate having the target substance, at which the first capturing molecule is immobilized on the surface of the substrate, is immersed in the evaluation liquid; The device for detecting a target substance, wherein the conductivity of the evaluation liquid before the substrate with the target substance is immersed therein is 1 mS / cm or less.

5. A substrate having a target substance and a sensor body are provided, The target substance-attached substrate includes a substrate having a first capture molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule in which a second capture molecule and an enzyme are bound to each other, and the second capture molecule is bound to the target substance; the sensor body includes an evaluation liquid containing a substrate to be modified by the enzyme, a container for accommodating the evaluation liquid, and an electric sensor provided to come into contact with the evaluation liquid for sensing a component in the evaluation liquid; the target substance-bearing substrate is independent and separate from the sensor body; a portion of the substrate having the target substance, at which the first capturing molecule is immobilized on the surface of the substrate, is immersed in the evaluation liquid; the electrical sensor is a FET sensor; An apparatus for detecting a target substance, wherein an ion-sensitive membrane is provided on the surface of the FET sensor.

6. 6. The device for detecting a target substance according to claim 2, 3, 4 or 5, wherein a part of the target substance-bearing substrate protrudes from the evaluation liquid.

7. 6. The device for detecting a target substance according to claim 1, 2, 3, 4 or 5, wherein the substrate is urea and the enzyme is urease.

8. 5. The device for detecting a target substance according to claim 1, 2, 3 or 4, wherein the electrical sensor is any one of an FET sensor, a conductivity sensor, an ion electrode and an impedance sensor.

9. The target substance detection device according to claim 1 , 2 , 3 , 4 , or 5 , wherein the electrical sensor is an FET sensor that uses graphene as a semiconductor channel.

10. The target substance detection device according to claim 2, 3, 4 or 5, wherein the substrate on which the first capture molecule is immobilized is composed of a magnetic particle on which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particle by magnetism.

11. A step of preparing a target substance-attached substrate, the target substance comprising a substrate having a first capturing molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capturing molecule, and an enzyme-labeled second capturing molecule in which a second capturing molecule and an enzyme are bound to each other and the second capturing molecule is bound to the target substance; immersing a portion of the substrate with the target substance, where the first capturing molecule is immobilized on the surface of the substrate, in an evaluation liquid containing a substrate to be modified by the enzyme; and sensing components in the evaluation liquid using an electrical sensor provided so as to be in contact with the evaluation liquid; A method for detecting a target substance, wherein the container is divided into a plurality of wells, each of which holds the evaluation liquid in a volume of 1 pL or less.

12. A step of preparing a target substance-attached substrate, the target substance comprising a substrate having a first capturing molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capturing molecule, and an enzyme-labeled second capturing molecule in which a second capturing molecule and an enzyme are bound to each other and the second capturing molecule is bound to the target substance; immersing a portion of the substrate with the target substance, where the first capturing molecule is immobilized on the surface of the substrate, in an evaluation liquid containing a substrate to be modified by the enzyme; and sensing components in the evaluation liquid using an electrical sensor provided so as to be in contact with the evaluation liquid; The end of the substrate is divided into two or more parts, The first capturing molecule is immobilized on at least one end of the substrate, The first capturing molecule is not immobilized on at least one end of the substrate, A method for detecting a target substance, wherein each end of the substrate is immersed in a different evaluation liquid.

13. A step of preparing a target substance-attached substrate, the target substance comprising a substrate having a first capturing molecule immobilized on at least a portion of a surface thereof, a target substance bound to the first capturing molecule, and an enzyme-labeled second capturing molecule in which a second capturing molecule and an enzyme are bound to each other and the second capturing molecule is bound to the target substance; immersing a portion of the substrate with the target substance, where the first capturing molecule is immobilized on the surface of the substrate, in an evaluation liquid containing a substrate to be modified by the enzyme; and sensing components in the evaluation liquid using an electrical sensor provided so as to be in contact with the evaluation liquid; The target substance includes a first target substance and a second target substance, The end of the substrate is divided into two or more parts, The first capturing molecule is immobilized on at least one end of the substrate, a third capturing molecule having a binding target different from that of the first capturing molecule is immobilized on at least one end of the substrate; The first target substance is bound to the first capturing molecule, The second target substance is bound to the third capturing molecule, the target substance-attached substrate further includes an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound to the second target substance, and the fourth capture molecule is bound to the second target substance; A method for detecting a target substance, wherein each end of the substrate is immersed in a different evaluation liquid.

14. The method for detecting a target substance according to claim 12 or 13, wherein the substrate carrying the target substance is immersed in the evaluation liquid such that a part of the substrate carrying the target substance protrudes from the evaluation liquid.

15. The method for detecting a target substance according to claim 11, 12 or 13, wherein the substrate with the target substance is prepared by immersing the substrate, on at least a portion of whose surface the first capture molecule is immobilized, in a preparation liquid containing the enzyme-labeled second capture molecule and the target substance.

16. The method for detecting a target substance according to claim 11, 12 or 13, wherein the substrate with the target substance is prepared by immersing the substrate, in which the first capture molecule is immobilized on at least a portion of the surface thereof and the target substance is bound to the first capture molecule, in a preparation liquid containing the enzyme-labeled second capture molecule.

17. The method for detecting a target substance according to claim 12 or 13, wherein the substrate to which the first capture molecule is immobilized is composed of a magnetic particle to which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particle by magnetism.

18. The method for detecting a target substance according to claim 11, 12 or 13, wherein the electrical sensor is any one of an FET sensor, a conductivity sensor, an ion electrode and an impedance sensor.